Rapid and specific detection of biological cells and biomolecules, such as red blood cells, white blood cells, platelets, proteins, DNA, and RNA, has become more and more important to biological assays that form a crucial element in diverse fields such as genomics, proteomics, diagnoses, and pathological studies. For example, the rapid and accurate detection of specific antigens and viruses is critical for combating pandemic diseases such as AIDS, flu, and other infectious diseases. Also, due to faster and more specific methods of separating and detecting cells and biomolecules, the molecular-level origins of diseases are being elucidated at a rapid pace, potentially ushering in a new era of personalized medicine in which a specific course of therapy is developed for each patient. To fully exploit this expanding knowledge of disease phenotype, new methods for detecting multiple biomolecules (e.g. viruses, DNA and proteins) simultaneously are required. Such multiplex biomolecule detection methods must be rapid, sensitive, highly parallel, and ideally capable of diagnosing cellular phenotype.
One specific type of biological assay increasingly used for medical diagnostics, as well as in food and environmental analysis, is the immunoassay. An immunoassay is a biochemical test that measures the level of a substance in a biological liquid, such as serum or urine, using the reaction of an antibody and its antigen. The assay takes advantage of the specific binding of an antibody to its antigen. Monoclonal antibodies are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules. The antibodies picked must have a high affinity for the antigen (if there is antigen in the sample, a very high proportion of it must bind to the antibody so that even when only a few antigens are present, they can be detected). In an immunoassay, either the presence of antigen or the patient's own antibodies (which in some cases are indicative of a disease) can be measured. For instance, when detecting infection the presence of antibody against the pathogen is measured. For measuring hormones such as insulin, the insulin acts as the antigen. Conventionally, for numerical results, the response of the fluid being measured is compared to standards of a known concentration. This is usually done though the plotting of a standard curve on a graph, the position of the curve at a response of the unknown is then examined, and so the quantity of the unknown found. The detection of the quantity of antibody or antigen present can be achieved by either the antigen or antibody.
An increasing amount of biological assays, such as immunoassays and gene expression analysis, are carried out using microarrays, such as DNA microarrays, protein microarrays or antibody microarrays, for example. A microarray is a collection of microscopic spots such as DNA, proteins or antibodies, attached to a substrate surface, such as a glass, plastic or silicon, and which thereby form a “microscopic” array. Such microarrays can be used to measure the expression levels of large numbers of genes or proteins simultaneously. The biomolecules, such as DNA, proteins or antibodies, on a microarray chip are typically detected through optical readout of fluorescent labels attached to a target molecule that is specifically attached or hybridized to a probe molecule. The labels used may consist of an enzyme, radioisotopes, or a fluorophore.
A large number of assays use a sandwich assay format for performing the assay. In this format, a capture probe molecule is immobilized on a surface. In the subsequent steps, a sample solution containing target molecules, also called analytes is applied to the surface. The target or analyte binds in a concentration dependent manner to the capture probe molecules immobilized on the surface. In a subsequent step, a solution containing detection probe molecules is applied to the surface, and the detection probe molecules can then bind to the analyte molecule. The analyte is thus “sandwiched” between the capture probe and detection probe molecules. In some assays, a secondary probe molecule is also applied to the assay, which can bind the detection probe molecule. The secondary probe can be conjugated to a fluorophore, in which case the binding result can be detected using a fluorescence scanner or a fluorescence microscope. In some cases, the secondary probe is conjugated to radioactive element, in which case the radioactivity is detected to read out the assay result. In some cases, the secondary probe is conjugated to an enzyme, in which case a solution containing a substrate has to be added to the surface, and the conversion of the substrate by the enzyme can be detected. The intensity of the signal detected is in all cases proportional to the concentration of the analyte in the sample solution.
Another type of cell and biomolecule separation and detection method uses microfluidic devices to conduct high throughput separation and analysis based on accurate flow controls through the microfluidic channels. By designing patterned fluidic channels, or channels with specific dimensions in the micro or sub-micro scales, often on a small chip, one is able to carry out multiple assays simultaneously. The cells and biomolecules in microfluidic assays are also typically detected by optical readout of fluorescent labels attached to a target cell or molecule that is specifically attached or hybridized to a probe molecule.
However, for protein analysis it remains very challenging to develop multiplexed assays. A number of recent attempts have been made to develop improved multiplexed antibody microarrays for use in quantitative proteomics, and various researchers have begun to examine the particular issues involved. Some of the general considerations in assembling multiplexed immunoassays have been found to include: requirements for elimination of assay cross-reactivity; configuration of multianalyte sensitivities; achievement of dynamic ranges appropriate for biological relevance when performed in diverse matrices and biological states; and optimization of reagent manufacturing and chip production to achieve acceptable reproducibility. In contrast to traditional monoplex enzyme-linked immunoassays, generally agreed specifications and standards for antibody microarrays have not yet been formulated.
The challenge of multiplexed immunoassay is further compounded when using complex biological samples, such as blood and its plasma and serum derivatives or other bodily fluids. The dynamic range of concentration of protein in blood has been found to span 11 orders of magnitude. Thus, when identifying low abundance proteins in blood, it has to be made against a background of proteins 11 orders of magnitude more numerous. As an analogy, if we were to identify a single person among the entire world population it would correspond to less than 10 orders of magnitude, as the world population is still less than 10 billion people.
Immunoassays and other assays exploiting microarrays exploit microfluidics. Microfluidics is concerned with handling and manipulating minute amounts of reagents. A major challenge in microfluidics is the mismatch between conventional liquid handling systems and the small scale of microfluidics, which constitutes a major obstacle to the more widespread adoption of microfluidics in laboratories and clinical settings, and has been described as the “world-to-chip” interface. The difficulty lies in delivering solutions from macroscopic containers such as vials or microplates to the microscopic reservoirs and channels of microfluidics rapidly, and without wastage. The interfacing problem becomes particularly challenging when large numbers of reagents need to be delivered to a microchip. Complex integrated microfluidic circuits have been built using so called multilayer soft lithography, see for example J. M. K. Ng et al in “Components for Integrated Poly(dimethylsiloxane) Microfluidic Systems” (Electrophoresis, Vol. 23, pp 3461-3473), but the delivery of reagents remains cumbersome, and often large external reservoirs with dead volumes are used, multiple tubings need to be manually connected, and reagent loading remains serial, all of which contribute to limit the versatility of these technologies. Many microfluidic chips are still loaded manually using pipettes which is slow, and with a lower limit for the volume of approximately 200 nl, but with little dead volume on the other hand, see for example L. Gervais et al in “Toward One-Step Point-of-Care Immunodiagnostics using Capillary-Driven Microfluidics and PDMS Substrates” (Lab on a Chip, Vol. 9, pp 3330-3337).
Microarrays although typically considered apart from microfluidics also depend on the transfer of minute amounts of reagents. In microarrays, the macro-to-micro challenge was addressed using large number of pins to transfer minute amount of liquids from microtiter plates to chips by repeatedly printing them onto multiple chips to minimize waste. The upload and transfer are controlled by capillary effects that need to be precisely engineered, see for example R. A. George et al in ““Ceramic Capillaries for use in Microarray Fabrication” (Genome Res., Vol. 11, pp 1780-1783) and R. Safavieh et al in “Straight SU-8 Pins” (J. Micromechanics and Microengineering, Vol. 20, 055001, 2010). Inkjet spotters with front-loading have also been developed and used to produce microarrays, see for example H. Li et al in “Hydrogel Droplet Microarrays with Trapped Antibody-Functionalized Beads for Multiplexed Protein Analysis” (Lab on a Chip, Vol. 11, pp 528-534) and M. Pia-Roca et al in “Addressable Nanowell Arrays Formed Using Reversibly Sealable Hybrid Elastomer-Metal Stencils” (Anal. Chem., Vol. 82, pp 3848-3855). The number of nozzles is typically much lower than that for pin spotters, however the programmability and rapid dispensing of droplets on-the-fly compensates for the limited parallelism. More recently, a novel system named the top spot has been presented which is made of a spotting head that is filled using capillary forces and for which dispensing is effected by compression of air above the nozzles, see for example C. Steinert et al in “TopSpot™ Vario: A Novel Microarrayer System for Highly Flexible and Highly Parallel Picoliter Dispensing” (Biomed. Microdevices, Vol. 11, 755-761). This system is overall simpler than inkjet spotters, but lacks individual addressing of the nozzles and requires larger volumes for loading the head. All these systems however remain reliant on robotics and are quite complex.
Recently, several groups proposed novel approaches to transfer minute amounts of reagents by using a “storage chip”. In this way, an array can first be formed on one or several chips using high precision inkjet spotters, and subsequently all reagents transferred to another chip, or mixed with a sample, at once. Du, Ismagilov and colleagues have developed an elegant approach called the “SlipChip”. With a “SlipChip”, nanoliter droplets of reagents are first trapped in channels and recesses which serve as reaction chambers, then a sample is loaded in a microchannel running parallel to the recesses, and then both are merged by sliding the two microstructured chips, see W. Du et al in “SlipChip” (Lab on a Chip, Vol. 9, 2286-2292).
To date, “SlipChips” have been used to deliver a single sample to an array of reagents, such as the delivery of single sample to 48 crystallization wells or to different chambers for sandwich immunoassays, see Du and W. Liu et al in “SlipChip for Immunoassays in Nanoliter Volumes” (Anal. Chem., Vol. 82, pp. 3276-3282), these examples represent a 1-to-N transfer. Alternative chip-to-chip transfer methods based on reagent diffusion from sol-gels and hydrogel spots have recently been proposed in the context of cell-based drug screening. First, the transfer of drugs and drug metabolites from sol-gel spots to cell monolayers on a flat substrate was demonstrated by M. Y. Lee et al in “Metabolizing Enzyme Toxicology Assay Chip (MetaChip) for High-Throughput Microscale Toxicity Analyses” (Proc. Natl. Acad. Sci. U.S.A., Vol. 102, pp. 983-987) and then the transfer from alginate gel droplets to cells encapsulated in collagen by T. G. Fernandes et al in “Three-Dimensional Cell Culture Microarray for High-Throughput Studies of Stem Cell Fate” (Biotechnol. and Bioeng., Vol. 106, pp. 106-118) and M-Y. Lee et al in “Three-dimensional Cellular Microarray for High-Throughput Toxicology Assays” (Proc. Natl. Acad. Sci. U.S.A, Vol. 105, pp. 59-63). More recently, Khademhosseini and colleagues adopted a similar approach to transfer drugs from approximately 200 μm wide posts made of either PDMS in “A Sandwiched Microarray Platform for Benchtop Cell-Based High Throughput Screening” (Biomaterials, Vol. 32, pp. 841-848) or a hydrogel in “Drug-Eluting Microarrays for Cell-Based Screening of Chemical-Induced Apoptosis” (Anal. Chem., Vol. 83, pp. 4118-4125) that were coated or loaded, respectively, with a drug library by inkjet spotting. The library was delivered at once to an array of 400 μm wide micro-wells on a microscope slide by clamping the chips and letting the drug diffuse into the buffer contained in each well. The wells were seeded with cells from a single cell line. This approach allowed selective delivery of a single drug per well, however a minor misalignment persisted possibly due to shrinkage of the PDMS. In summary, for the chip transfer methods described to date, manual alignment based on visible structures on the chip was used, and the transfer followed an N-to-1 or a 1-to-N arrangement with N different reagents being reacted or mixed with a single kind of sample.
In conventional multiplexed sandwich assays in both array and bead formats, the detection antibodies are applied as a mixture, which is much simpler than multi-spotting, but gives rise to interactions among reagents that each constitute a liability for cross-reactivity, which in turn entails lengthy and costly optimization protocols and which severely limits the performance of these assays. Recently, we proposed the antibody colocalization microarray (ACM), see M. Pla-Roca et al in “Antibody Colocalization Microarray: A Scalable Method for Multiplexed and Quantitative Protein Profiling” (submitted to Mol. Cell. Proteomics), which depends on the addressing of each capture antibody spot by a single detection antibody, thus colocalizes each pair and reproducing assay conditions that are found in single-plex ELISA assays, but only requires less than a nanoliter of antibody reagents. The execution of an ACM requires first spotting the capture antibody, removing the slide from the spotter, incubating it with sample, washing and rinsing it as needed, and placing it back for the spotting of the detection antibody followed by binding and incubation. ACM depends on the transfer of N different reagents to N spots each with a different reagent as well, representing an N-to-N transfer. Local addressing was achieved using a custom built microarrayer with precise alignment mechanisms, but unlike approaches with mixing of reagents, spotting needs to be performed as part of the assay, which is cumbersome, and constitutes an obstacle to the adoption of ACM by others.
Here, we present the snap chip for the collective transfer of a microarray of reagents contained within semi-spherical liquid droplets to a target microarray following assembly of the two chips and physical contact of the droplets with the target array. Nanoliters of reagents are spotted on both slides using an inkjet spotter, and selectively transferred from liquid droplets on a transfer chip to an assay chip within the contact areas. A process with back-side alignment and a hand-held snap apparatus were developed to allow for simple and reliable transfer of reagents of an entire microarray. Using the snap chip, we performed multiplexed sandwich immunoassays with colocalization of capture and detection antibodies with 10 targets simultaneously with detection limits in the low pg/ml in buffer and in 10% serum. Finally, we established a protocol for long term storage, three month in this study, of both the assay and transfer chips.